Considerable evidence in animal models and in humans indicates that bone loss in periodontitis is produced by osteoclasts. The origin of osteoclasts and the factors responsible for their recruitment, differentiation, and activation are not well defined. A newly described, required factor for the differentiation and activation of osteoclasts is Receptor and Activator of NF-kB Ligand (RANKL) which mediates its effects via a specific cell surface receptor, Receptor and Activator of NF-kB (RANK). This receptor is a member of the TNF receptor family, and is expressed on osteoclasts and osteoclast precursors. With RANKL binding to its receptor, a cascade of signal transduction events occurs to ultimately culminate in the activation of transcription factors which effect expression of target genes important for osteoclast differentiation. NF-kB is one such transcription factor known to be activated and required for osteoclastogenesis. The signaling pathway from receptor to activation of NF-kB is not understood. The intracellular domain of RANK is linked to downstream signaling molecules by adapter proteins of the TNF Receptor Associated Factor (TRAF) family, which have no intrinsic catalytic properties, but are able to associate with molecules that do. One member, TRAF6, associates with RANK, and links it to a MAF3 kinase named TGFbeta Activated Kinase1 (TAK1), which is involved in NF-kB activation. The studies outlined in this proposal will examine how TRAF6 functions to activate TAK1 and induce NF-kB activation and how these events lead to osteoclast differentiation and activation. Aim 1 will test the hypothesis that RANK activation of NF-kB is mediated by a TAK1 dependent signaling pathway. First, TAK1 interaction with TRAF6 will be examined and the interaction regions mapped . In addition, the importance of the kinase function of TAK1 for signaling will be defined. Involvement of a likely downstream target of TAK1 will be examined and the effect of TAK1 inhibition on osteoclast differentiation assessed. Specific Aim 2 will test the hypothesis that the function of the Ring and Zinc Finger (RZF) domain of TRAF6 may involve interaction with TAB2 to accomplish cell membrane localization. Involvement of the different domains of TRAF6 in membrane localization and NF-kB activation will be examined in light of a newly reported membrane associated signaling intermediate, TAB2. A more detailed understanding of the basic regulation of osteoclastogenesis may permit development of potent novel therapeutics for treating periodontal disease and other inflammatory disorders associated with bone loss.